M. Isani1, A. Grishin1, H. Ford1 1Children’s Hospital Los Angeles,Los Angeles, CA, USA
Introduction:
Necrotizing enterocolitis (NEC) affects premature infants and its etiology remains largely unknown. Colonization of the neonatal gastrointestinal (GI) tract with opportunistic pathogens, such as Coronobacter muytjensii, is thought to be a prime initiating event. Opportunistic pathogens compromise the gut barrier, leading to bacterial translocation, inflammation, and, ultimately, intestinal necrosis. Lactobacillus biotics have been shown in a number of studies to protect against NEC. However, results of Lactobacillus trials remain inconclusive due to the use of different species/strains and doses in different trials. Moreover, an important question of whether the lactobacilli used as probiotics are colonizing the intestine remains unanswered. We propose that an efficient probiotic strain should not only protect the intestinal epithelium, but should also be capable of colonizing the GI tract. To identify naturally occurring colonizing lactobacilli, we sought to isolate and characterize the strains of this genus in the intestines of 4 day old rats.
Methods:
Animal experiments were approved by CHLA IACUC. Neonates were obtained from timed pregnant Sprague Dawley rats purchased from Charles River or Harlan Labs. The neonates were kept in a temperature and humidity controlled incubator, and fed with formula for 4 days. Care was taken not to introduce extraneous bacteria during handling and feeding. To enumerate and isolate the lactobacilli, the content of the large intestine was serially diluted and plated on MRS agar. After 2 day incubation at 37oC, the emerging colonies were classified according to their appearance, and numbers in each class counted. Pure cultures were established for each class and maintained as frozen stocks. The lactobacilli were identified as Gram+ or Gram+/- non-spore-forming rods. The species identity was established by sequencing a variable region of the 16S rRNA gene and by biochemical tests.
Results:
Seven morphologically unique strains of lactobacilli were isolated from 138 rat pups belonging to 18 litters (Figure 1). Among the identified species, there were L. reuteri and L. murinus.
Conclusion:
Various lactobacilli are common first GI tract colonizers in neonatal rats. Availability of the pure cultures of the natural colonizing Lactobacillus strains will allow us to experimentally evaluate their ability to colonize the intestine and protect against NEC.
